High-Throughput System for the Presentation of Secreted and Surface-Exposed Proteins from Gram-Positive Bacteria in Functional Metagenomics Studies

Complex microbial ecosystems are increasingly studied through the use of metagenomics approaches. Overwhelming amounts of DNA sequence data are generated to describe the ecosystems, and allow to search for correlations between gene occurrence and clinical (e.g. in studies of the gut microbiota), physico-chemical (e.g. in studies of soil or water environments), or other parameters. Observed correlations can then be used to formulate hypotheses concerning microbial gene functions in relation to the ecosystem studied. In this context, functional metagenomics studies aim to validate these hypotheses and to explore the mechanisms involved. One possible approach is to PCR amplify or chemically synthesize genes of interest and to express them in a suitable host in order to study their function. For bacterial genes, Escherichia coli is often used as the expression host but, depending on the origin and nature of the genes of interest and the test system used to evaluate their putative function, other expression systems may be preferable. In this study, we developed a system to evaluate the role of secreted and surface-exposed proteins from Gram-positive bacteria in the human gut microbiota in immune modulation. We chose to use a Gram-positive host bacterium, Bacillus subtilis, and modified it to provide an expression background that behaves neutral in a cell-based immune modulation assay, in vitro. We also adapted an E. coli – B. subtilis shuttle expression vector for use with the Gateway high-throughput cloning system. Finally, we demonstrate the functionality of this host-vector system through the cloning and expression of a flagellin-coding sequence, and show that the expression-clone elicits an inflammatory response in a human intestinal epithelial cell line. The expression host can easily be adapted to assure neutrality in other assay systems, allowing the use of the presented presentation system in functional metagenomics of the gut and other ecosystems.     

Associations between Lifetime Traumatic Events and Subsequent Chronic Physical Conditions: A Cross-National,Cross-Sectional Study

Background

Associations between lifetime traumatic event (LTE) exposures and subsequent physical ill-health are well established but it has remained unclear whether these are explained by PTSD or other mental disorders. This study examined this question and investigated whether associations varied by type and number of LTEs, across physical condition outcomes, or across countries.

Methods

Cross-sectional, face-to-face household surveys of adults (18+) were conducted in 14 countries (n = 38, 051). The Composite International Diagnostic Interview assessed lifetime LTEs and DSM-IV mental disorders. Chronic physical conditions were ascertained by self-report of physician''s diagnosis and year of diagnosis or onset. Survival analyses estimated associations between the number and type of LTEs with the subsequent onset of 11 physical conditions, with and without adjustment for mental disorders.

Findings

A dose-response association was found between increasing number of LTEs and odds of any physical condition onset (OR 1.5 [95% CI: 1.4–1.5] for 1 LTE; 2.1 [2.0–2.3] for 5+ LTEs), independent of all mental disorders. Associations did not vary greatly by type of LTE (except for combat and other war experience), nor across countries. A history of 1 LTE was associated with 7/11 of the physical conditions (ORs 1.3 [1.2–1.5] to 1.7 [1.4–2.0]) and a history of 5+ LTEs was associated with 9/11 physical conditions (ORs 1.8 [1.3–2.4] to 3.6 [2.0–6.5]), the exceptions being cancer and stroke.

Conclusions

Traumatic events are associated with adverse downstream effects on physical health, independent of PTSD and other mental disorders. Although the associations are modest they have public health implications due to the high prevalence of traumatic events and the range of common physical conditions affected. The effects of traumatic stress are a concern for all medical professionals and researchers, not just mental health specialists.     

Alcohol Drinking Cessation and the Risk of Laryngeal and Pharyngeal Cancers: A Systematic Review and Meta-Analysis

Objective

To evaluate the effect of alcohol cessation on the risk of developing laryngeal and pharyngeal cancers, combining available evidence in the scientific literature in a meta-analysis.

Methods

A systematic literature review was conducted, and a meta-analysis was applied on the retrieved studies. The generalised least squares method was used to estimate the trend from dose-response data to assess changes in the risks of laryngeal and pharyngeal cancers after drinking cessation.

Results

A total of 9 case-control studies were included in the meta-analysis (4 and 8 estimates for laryngeal and pharyngeal cancers, respectively). On average, alcohol drinking cessation was associated with a 2% yearly reduction in the risk of developing laryngeal and pharyngeal cancers. There was a considerable heterogeneity between the studies of pharyngeal cancer, but this was mostly due to two studies. The increased risk of laryngeal and pharyngeal cancers caused by alcohol was reversible; the time periods until the risks became equal to those of never drinkers were 36 (95% CI 11–106) and 39 (95% CI 13–103) years, respectively. Moreover, 5 years of drinking cessation was associated with a reduction of around 15% in the alcohol-related elevated risk of laryngeal and pharyngeal cancers.

Conclusion

Although a long time period is required to completely eliminate the alcohol-related elevated risk of laryngeal and pharyngeal cancers, a substantial risk reduction can be seen in the short term (5–10 years), and drinking cessation should therefore be encouraged to reduce the incidence of these cancers.     

An Evaluation of Plotless Sampling Using Vegetation Simulations and Field Data from a Mangrove Forest

In vegetation science and forest management, tree density is often used as a variable. To determine the value of this variable, reliable field methods are necessary. When vegetation is sparse or not easily accessible, the use of sample plots is not feasible in the field. Therefore, plotless methods, like the Point Centred Quarter Method, are often used as an alternative. In this study we investigate the accuracy of different plotless sampling methods. To this end, tree densities of a mangrove forest were determined and compared with estimates provided by several plotless methods. None of these methods proved accurate across all field sites with mean underestimations up to 97% and mean overestimations up to 53% in the field. Applying the methods to different vegetation patterns shows that when random spatial distributions were used the true density was included within the 95% confidence limits of all the plotless methods tested. It was also found that, besides aggregation and regularity, density trends often found in mangroves contribute to the unreliability. This outcome raises questions about the use of plotless sampling in forest monitoring and management, as well as for estimates of density-based carbon sequestration. We give recommendations to minimize errors in vegetation surveys and recommendations for further in-depth research.     

Neighborhood Ethnic Diversity and Behavioral and Emotional Problems in 3 Year Olds: Results from the Generation R Study

Background

Studies suggest that neighborhood ethnic diversity may be important when it comes to understanding ethnic inequalities in mental health. The primary aim of this study was to investigate whether neighborhood ethnic diversity moderated the association between the ethnic minority status and child behavioral and emotional problems.

Methods

We included 3076 preschoolers participating in the Generation R Study, a birth cohort study in Rotterdam, the Netherlands. At child age 3-years, parents completed the Child Behavior Checklist (CBCL/1,5-5). Individual-level data, assessed with questionnaires, was combined with neighborhood-level data. Multi-level logistic regression models predicted the Odds Ratios for the CBCL total problems score as a function of maternal ethnic background and neighborhood ethnic diversity, computed with the Racial Diversity Index and categorized into tertiles. Interaction on the additive scale was assessed using Relative Access Risk due to Interaction.

Results

Being from an ethnic minority was associated with child behavioral and emotional problems in unadjusted (OR 2.76, 95% CI 1.88–4.04) and adjusted models (OR 2.64, 95% CI 1.79–3.92). Residing in a high diversity neighborhood was associated with child behavioral and emotional problems in unadjusted (OR 2.03, 95% CI 1.13–3.64) but not in adjusted models (OR 0.89, 95% CI 0.51–1.57). When stratifying by the three levels of neighborhood ethnic diversity, ethnic inequalities in behavioral and emotional problems were greatest in low diversity neighborhoods (OR 5.24, 95%CI 2.47–11.14), smaller in high diversity neighborhoods (OR 3.15, 95% CI 1.66–5.99) and smallest in medium diversity neighborhoods (OR 1.59, 95% CI 0.90–2.82). Tests for interaction (when comparing medium to low diversity neighborhoods) trended towards negative on both the additive and multiplicative scale for the maternal-report (RERI: −3.22, 95% CI −0.70–0.59; Ratio of ORs: 0.30, 95% CI 0.12–0.76).

Conclusion

This study suggests that ethnic inequalities in child behavioral and emotional problems may be greatest in ethnically homogeneous neighborhoods.     

Child Behavior Checklist—Mania Scale (CBCL-MS): Development and Evaluation of a Population-Based Screening Scale for Bipolar Disorder

Context

Early identification of Bipolar Disorder (BD) remains poor despite the high levels of disability associated with the disorder.

Objective

We developed and evaluated a new DSM orientated scale for the identification of young people at risk for BD based on the Child Behavior Checklist (CBCL) and compared its performance against the CBCL-Pediatric Bipolar Disorder (CBCL-PBD) and the CBCL-Externalizing Scale, the two most widely used scales.

Methods

The new scale, CBCL-Mania Scale (CBCL-MS), comprises 19 CBCL items that directly correspond to operational criteria for mania. We tested the reliability, longitudinal stability and diagnostic accuracy of the CBCL-MS on data from the TRacking Adolescents'' Individual Lives Survey (TRAILS), a prospective epidemiological cohort study of 2230 Dutch youths assessed with the CBCL at ages 11, 13 and 16. At age 19 lifetime psychiatric diagnoses were ascertained with the Composite International Diagnostic Interview. We compared the predictive ability of the CBCL-MS against the CBCL-Externalising Scale and the CBCL-PBD in the TRAILS sample.

Results

The CBCL-MS had high internal consistency and satisfactory accuracy (area under the curve = 0.64) in this general population sample. Principal Component Analyses, followed by parallel analyses and confirmatory factor analyses, identified four factors corresponding to distractibility/disinhibition, psychosis, increased libido and disrupted sleep. This factor structure remained stable across all assessment ages. Logistic regression analyses showed that the CBCL-MS had significantly higher predictive ability than both the other scales.

Conclusions

Our data demonstrate that the CBCL-MS is a promising screening instrument for BD. The factor structure of the CBCL-MS showed remarkable temporal stability between late childhood and early adulthood suggesting that it maps on to meaningful developmental dimensions of liability to BD.     

The Choice of PCR Primers Has Great Impact on Assessments of Bacterial Community Diversity and Dynamics in a Wastewater Treatment Plant

Assessments of bacterial community diversity and dynamics are fundamental for the understanding of microbial ecology as well as biotechnological applications. We show that the choice of PCR primers has great impact on the results of analyses of diversity and dynamics using gene libraries and DNA fingerprinting. Two universal primer pairs targeting the 16S rRNA gene, 27F&1492R and 63F&M1387R, were compared and evaluated by analyzing the bacterial community in the activated sludge of a large-scale wastewater treatment plant. The two primer pairs targeted distinct parts of the bacterial community, none encompassing the other, both with similar richness. Had only one primer pair been used, very different conclusions had been drawn regarding dominant phylogenetic and putative functional groups. With 27F&1492R, Betaproteobacteria would have been determined to be the dominating taxa while 63F&M1387R would have described Alphaproteobacteria as the most common taxa. Microscopy and fluorescence in situ hybridization analysis showed that both Alphaproteobacteria and Betaproteobacteria were abundant in the activated sludge, confirming that the two primer pairs target two different fractions of the bacterial community. Furthermore, terminal restriction fragment polymorphism analyses of a series of four activated sludge samples showed that the two primer pairs would have resulted in different conclusions about community stability and the factors contributing to changes in community composition. In conclusion, different PCR primer pairs, although considered universal, target different ranges of bacteria and will thus show the diversity and dynamics of different fractions of the bacterial community in the analyzed sample. We also show that while a database search can serve as an indicator of how universal a primer pair is, an experimental assessment is necessary to evaluate the suitability for a specific environmental sample.     

Early Life Stress and Physical and Psychosocial Functioning in Late Adulthood

Background

Severe stress experienced in early life may have long-term effects on adult physiological and psychological health and well-being. We studied physical and psychosocial functioning in late adulthood in subjects separated temporarily from their parents in childhood during World War II.

Methods

The 1803 participants belong to the Helsinki Birth Cohort Study, born 1934–44. Of them, 267 (14.8%) had been evacuated abroad in childhood during WWII and the remaining subjects served as controls. Physical and psychosocial functioning was assessed with the Short Form 36 scale (SF-36) between 2001 and 2004. A test for trends was based on linear regression. All analyses were adjusted for age at clinical examination, social class in childhood and adulthood, smoking, alcohol intake, physical activity, body mass index, cardiovascular disease and diabetes.

Results

Physical functioning in late adulthood was lower among the separated men compared to non-separated men (b = −0.40, 95% confidence interval [95% CI]: −0.71 to −0.08). Those men separated in school age (>7 years) and who were separated for a duration over 2 years had the highest risk for lower physical functioning (b = −0.89, 95% CI: −1.58 to −0.20) and (b = −0.65, 95% CI: −1.25 to −0.05), respectively). Men separated for a duration over 2 years also had lower psychosocial functioning (b = −0.70, 95% CI: −1.35 to −0.06). These differences in physical and psychosocial functioning were not observed among women.

Conclusion

Early life stress may increase the risk for impaired physical functioning in late adulthood among men. Timing and duration of the separation influenced the physical and psychosocial functioning in late adulthood.     

THE COMPOSITION OF WATER COLLECTED FROM THE KUISEB RIVER,NAMIB DESERT,AT GOBABEB

SUMMARY

Water from the Kuiseb River at Gobabeb varied in ionic content. Various ionic dominance orders were recorded from flooding until the pools dried up. The quality was not suitable for irrigation, but would present no problems when used by stock or wildlife.